1. Field of the Invention
The subject invention relates to a set of DNA primers which, when utilized in conjunction with the polymerase chain reaction (PCR) assay, can amplify and speciate DNA from four medically important Candida species. Furthermore, the PCR amplified DNA products generated by the primers can be sequenced, and the sequence information can be used to create species-specific probes which can confirm these four species of Candida. Thus, the primers and probes of the present invention allow for earlier diagnosis and treatment of an infection compared to diagnosis and treatment resulting from the standard method of routine culturing.
2. Background Information
Diagnosing systemic Candida infection continues to be a major challenge for the clinician. This is especially true in the low birth weight (VLBW) neonate where, historically, 30% of all neonatal systemic Candidiasis is not diagnosed until autopsy ("Systemic Candidiasis" In Candidiasis, eds., Bodey et al., Raven Press, N.Y. (1985)). The difficulty in diagnosing neonatal candidemia is due, in part, to the lack of a sensitive detection system and to the minimal blood volume that can be obtained for testing. Currently, the gold standard for diagnosing systemic candidiasis is to recover the organism by routine blood culturing (Kiehn et al., J. Clin. Microbiol 14:681-83 (1981); Roberts et al., J. Clin. Microbiol. 1:309-10 (1975)). This technique is not always satisfactory in detecting infection early, a critical point for successful outcome in treating systemic candidiasis. In fact, 40-60% of all blood cultures remain negative for Candida despite widespread visceral infection ("Systemic Candidiasis" In Candidiasis, eds., Bodey et al., Raven Press, N.Y. (1985)).
Furthermore, in addition to the time required to grow the yeast from blood culture, additional time is necessary in which to identify and speciate the organism. All of this testing can become a very lengthy process, leaving the infection unchecked in the patient, leading to an increased fungal load.
Unlike the comparative safety of antibiotics, antifungal drugs are quite toxic. Therefore, physicians usually require substantial documentation of fungal infection before they are willing to initiate antifungal therapy. Prompt detection of candidiasis would enable earlier treatment initiation on a smaller fungal load. Thus, timely detection and speciation of Candida infections is crucial for reducing morbidity and mortality especially in the premature newborn and the immunosuppressed population (i.e., bone marrow transplant patients, AIDS patients, etc.).
It is the amplification property of the polymerase chain reaction (PCR) assay together with its short turnaround time that makes it ideal for diagnosing candidemia from even minimal blood volumes. PCR is an efficient, in-vitro method for amplifying DNA from clinical samples that can be tailor-made to suit the needs of any diagnostic laboratory (see U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,683,195). Buchman et al. was the first group to describe the use of PCR to identify Candida albicans from clinical specimens. These investigators used PCR to amplify a portion of a yeast-specific gene cytochrome lanosterol-14.alpha.-demethylase (Buchman et al., Surgery 108:338-47 (1990)).
More specifically, Buchman et al. utilized a set of primers that amplified the Candida albicans cytochrome P.sub.450 L.sub.1 A.sub.1 (lanosterol-14.alpha.-demethylase) gene. The predicted product size was estimated to be 240 base pairs. However, aberrant and unexplained amplification patterns were seen in various clinical specimens containing C. albicans DNA.
In addition, the primer set utilized by Buchman et al. amplified DNA from other non-albicans species giving rise to some PCR products of the "predicted" 240 bp size and some of an alternate size. Other non-albican species gave no 240 bp fragment but just a variety of alternate-sized products. There was no consistency in the size of the PCR products generated using this primer set.